FIG. 2.

Pop#8 identification by lineage exclusion. Applying multicolor flow cytometry of freshly isolated hBM-MNCs, events generated by single cells were selected on the basis of scatter signals and plotted on CD18 versus CD31 density plot. Seven regions were identified (R1–R7) according to a combination of four levels of CD31/CD18 intensity: Neg (negative), Dim (low level of expression), Int (intermediate level of expression), and Bright (high level of expression) (A). Gating on lineage-negative cells (not expressing CD3, CD20, CD14, or CD66), CD18 versus CD31 density plot revealed in all samples a residual CD31^brightCD18^dim/int population that we defined as unknown population #8 (Pop#8) (B). Backgating strategy showed that most of the well-acquainted hBM populations, such as CD105⁺ (yellow dots), MSCA-1⁺ (light green dots), mature lymphocytes (orange dots), granulocytes (light blue dots), HSCs (pink dots), and plasma cells (violet dots) occupied one or more of the expected R1–R7 regions (C). Conversely, Pop#8 (black dots) occupied one distinct region (R8). HSCs, hematopoietic stem cells.